Determination of gamma-glutamyl transpeptidase in biological fluids

ABSTRACT

A method for determining gamma-glutamyl transpeptidase in biological fluids is described which comprises incubating the test fluid with an aqueous buffered solution of an L-gammaglutamyl-p-nitroanilide substrate, glycylglycine, sodium nitrite, and optionally, magnesium chloride hexahydrate, for a predetermined period of time; adding, after incubation is completed, a mineral acid reagent solution of 8-hydroxy quinoline sulfate containing a water soluble, nonionic emulsifier, the mineral acid solution having a pH of between 0.8 and 1.3; adding an alkaline solution which will impart a pH of between 10.5 and 12, in the total reaction solution, said alkaline solution optionally containing ethylenediaminetetraacetic acid, tetrasodium salt; and reading the optical density of the total reaction solution as a measure of gamma-glutamyltranspeptidase concentration in the test sample of biological fluid. The method of the invention is accurate and approximately 4.5 times more sensitive than the standard test method currently being used.

United States Patent Carroll DETERMINATION OF GAMMA-GLUTAMYLTRANSPEPTIDASE IN BIOLOGICAL FLUIDS [21] Appl. No.: 282,632

OTHER PUBLICATIONS Glick, Methods of Biochemical Analysis Vol. 13 Pages347 & 348 (1965).

Primary Examiner-Alvin E. Tanenholtz Att0rneyAlbert H. Graddis etal.

[ Oct. 30, 1973 [57] ABSTRACT A method for determining gamma-glutamyltranspeptidase in biological fluids is described which comprisesincubating the test fluid with an aqueous buffered solution of anL-gamma-glutamyl-p-nitroanilide substrate, glycylglycine, sodiumnitrite, and optionally, magnesium chloride hexahydrate, for apredetermined period of time; adding, after incubation is completed, amineral acid reagent solution of 8-hydroxy quinoline sulfate containinga water soluble, nonionic emulsifier,

the mineral acid solution having a pH of between 0.8 and 1.3; adding analkaline solution which will impart a pH of between 10.5 and 12, in thetotal reaction solution, said alkaline solution optionally containingethylenediaminetetraacetic acid, tetrasodium salt; and reading theoptical density of the total reaction solution as a measure ofgamma-glutamyltranspeptidase concentration in the test sample ofbiological fluid. The method of the invention is accurate andapproximately 4.5 times more sensitive than the standard test methodcurrently being used.

11 SEE NPUPFEY V DETERMINATION OF GAMMA-GLUTAMYL TRANSPEPTIDASE INBIOLOGICAL FLUIDS BACKGROUND OF THE INVENTION gamma-Glutamyltranspeptidase is an enzyme found in kidney extracts which catalyzestranspeptidation reactions involving metabolic gamma-glutamyl peptidesand amino acids, wherein new peptides are formed containing thegamma-glutamyl moiety. The reaction consists in the transfer of thegamma-glutamyl group from linkage with the peptide to linkage with theamino acid group in an acceptor amino acid. Thus, transpeptide reactionsresult in a redistribution of gammaglutamyl groups.

gamma-Glutamyl transpeptidase is found primarily in the kidneys ofvarious animal species. Procedures are known for the isolation ofgamma-glutamyl transpeptidase from sheep kidneys, pig kidneys, oxkidneys and pancreas, as well as from kidneys of cows and humans.

gamma-Glutamyl transpeptidase is known to be present in various bodyorgans and tissues, as well as in urine and in blood serum and'plasma.Recently, the detection of abnormal levels of gamma-glutamyltranspeptidase in certain body fluids has been found to indicate thepresence of certain disease conditions. For example, Szczeklik et al.,Gastroenterology, 4l: 353-359 (1961) reported that only moderateincreases of gamma-glutamyl transpeptidase were observed in patientswith viral or chronic hepatitis. However, very high activities werefound in patients suffering from obstructive jaundice, biliarycirrhosis, cholangitis, and liver neoplasia, both primary liver cancerand intrahepatic metastases. The enzyme activity in these patients wasoccasionally 100 times as great as the activity in normal patients. Veryhigh gamma-glutamyl transpeptidase activity, not associated withjaundice, is highly suspicious of a primay or metastatic liver cancer.

In 1966, Nosslin et al., Scand. J. Clin. and Lab. In-

vest., 18: 178-80 Suppl. 92 (1966) compared gamma-' glutamyltranspeptidase activity with alkaline phosphatase in cases consisting ofhepatitis and obstructive jaundice. The authors concluded that althoughphosphatase and gamma-glutamyl transpeptidase had identical reactionpatterns, transpeptidase was more demonstrable in thepathologicalconditions studied.

Further, Lum et al., Clin. Chem. 18: 358-362 (1972) demonstrated thatgamma-glutamyl transpeptidase activity was above normal in all forms ofliver disease studied (viral hepatitis, cirrhosis, cholecystitis,metastatic carcinoma to liver, pancreaticcarcinoma, liver granuloma, andacute pancreatitis). gamma-Glutamyl transpeptidase more sensitivelyindicated hepatic disease than did alkaline phosphatase, much more sothan did leucine aminopeptidase. Moreover, it was found that measurementof gamma-glutamyl transpeptidase activity offered a simple, sensitive,and direct means for distinguishing whether bone or liver was the sourceof increased serum alkaline phosphatase activity. Activity was highestin obstructive liver disease.

Over the years, a great variety of methods have been described for thedetermination of gamma-glutamyl transpeptidase concentration inbiological fluids. Historically, glutathione was used as the substrateand the action of gamma-glutamyl transpeptidase on the substrate wasmeasured. More recently, synthetic substrates includinggamma-D,L-glutamylaminopropionitrile;

gamma-L-glutamyl-anapthylamide; and N-D,L-gamma-glutamyl-anilide havebeen utilized in such determinations. In these last mentioned assays,the appearance of one or more cleavage products resulting from theaction of the gamma-glutamyl transpeptidase in the biological fluidbeing tested on the substrate was considered a measure of enzymeactivity; improved test procedures resulted by reacting the cleavageproduct with a dye former in order to utilize photometricinstrumentation for measurements. A further simplification of thisgeneral test method resulted when gamma-L-glutamyl-pnitroanilide wasused as the substrate, since a colored cleavage product, i.e.,p-nitroaniline, resulted which could be measured colorimetricallywithout further reaction to form a dye.

Determination of gamma-glutamyl transpeptidase activity utilizinggamma-L-glutamyl-p-nitroanilide as the substrate and variousmodifications of this test method, have been investigated by a number ofscientists, including Orlowski, M., Arch. lmmunol. Therap. Exptl. 13:538 (1964); Dimov, D.M. et al., Clin. Chem. Act. 16: 271-277 (1967); andSzasz, 0., Clin. Chem. 15: 124-136 (1969).

Among the above mentioned methods for determining gamma-glutamyltranspeptidase activity, the Szasz procedure has gained widespreadacceptance and is now generally considered the standard clinical method.The chief advantage of the test is its great simplicity, for in routineclinical testing the omission or acceleration of any procedural steps isparticularly desirable. Nonetheless, there are still certaindisadvantages to the Szasz method. For example,a sample blank should berun for each serum sample to assure accurate'determinations, sincesample turbidity often interferes with optical density measurements. Inaddition, the Szasz method does not take into account non-enzymatichydrolysis of the substrate; this necessarily limits the accuracy ofdeterminations, due to the fact that high levels of enzymatic activityin any sample being tested could yield optical density readings beyondthe range of the usual clinical laboratory instruments. This problem isfurther compounded in situations where very high levels of bilirubin,which imparts a yellow coloration, are present in the test sample: theyellow color is amplified to the extent that more sophisticatedinstruments are required for accurate measurements.

For all of the above reasons, automation of the Szasz method would bemost difficult.

Thus, there is a real need for an improved method for the determinationof gamma-glutamyl transpeptidase concentration in body fluids.

SUMMARY OF THE INVENTION taining a water-soluble, nonionic emulsifier,which mineral acid solution has a pH of from 0.8 to L3; after the elapseof 15 seconds to 10 minutes, a sufficient amount of an alkaline solutionis added to impart a pH of from 10.5 to 12 to the reaction solution,whereby there is obtained a differently colored reaction solution, theabsorbence of which provides a sensitive, ac curate measure ofgamma-glutamyl transpeptidase con centration in the biological fluidbeing tested.

DESCRIPTION OF THE INVENTION A sensitive, accurate method for thedetermination of gamma-glutamyl transpeptidase activity in biologicalfluids has now been developed. According to the method of thisinvention, the biological test fluid is incubated for a pre-determinedperiod of time, with a buffered solution containing p-nitroanilidesubstrate, glycylglycine, and sodium nitrite to obtain a pnitroanilinecleavage product in the reactionsolution. This reaction solution isfurther treated with an acid solution of 8-hydroxy quinoline sulfate,containing an emulsifier, at a controlled pH. After the elapse ofseconds to 10 minutes, a sufficient amount of alkali is added to bringthe reaction solution to an alkaline pH.

The optical density of the purple colored reaction solution obtained isthen read as a measure of enzyme activity in the biological test fluid.

The novel assay of the invention has been found to be four and one-halftimes more sensitive than the prior art Szasz method. Thus, there isprovided a simple, accurate, and sensitive method for determining enzymeconcentration without the necessity of running blanks for each samplebeing tested. Moreover, the bilirubin normally present in body fluids nolonger has an adverse effect on the absorbency readings. The opticaldensity of the purple-colored solution of the cleavage product can beread directly against a standardcurve of the absorbence of freep-nitroaniline solutions, subjected to the same dye-forming reactions.The activity of the gamma-glutarn'yl transpeptidase enzyme is expressedin International Units (I.U.) as the amount of micromoles ofp-nitroaniline released per 1,000 ml of serum in one minute.

' The substrate used in the assay of this'invention is a bufferedsolution of L-gam ma-glutamyl-p-nitroanilide', giycyl-glycine, sodiumnitrite and, optionally, magnesium chloride hexahydrate. TheL-gamma-glutamyl-pnitroanilide is thesubstrate acted upon by thegammaglutamyl transpeptidase enzyme. The glycylglycine acts as anacceptor for the glutamyl moiety. The sodium nitrite, in an acidenvironment, diazotizes the pnitroaniline nitroaniline cleavage product.The magnesium chloride hexahydrate, which is an optional ingredient, isthought to aid in keeping the substrate in solution and is alsoconsidered to activate the reaction. However, the use of magnesiumchloride hexahydrate is not absolutely necessary in the diagnostic testof the invention.

The buffer in the substrate solution maintains the solution at a pH offrom about 7.9 to about 8.4, preferably about 8.1, for optimum enzymaticactivity. As the buffer, there may be used (2-amino-2-methyl-propane-1,3-dio1)-hydrochloride (known as ammediol-HCI), andtris(hydroxymethyl)amino methane hydrochloride (known as Tris-HCl).

The amount of substrate, glycylglycine, sodium nitrite, and magnesiumchloride hexahydrate required for the diagnostic test of this inventionis dependent upon the amount of biological fluid being tested. It hasbeen determined that the buffered substrate solution should contain atleast 45 micromoles of L-gamma-glutamylp-nitroanilide, at least 400micromoles of glycyiglycine, at least 36.2 micromoles of sodium nitrite,and, optionally, at least micromoles of magnesium chloride hexahydrateper milliliter of biological fluid in order to insure that a sufficientamount of reagents is present for reaction with the highest known levelof enzyme in the biological fluid being tested. For the sodium nitritereagent, the minimum amount required is, as mentioned above, at least36.2 micromoles per milliliter of biological fluid. However, it isgenerally advantageous to have an excess of sodium nitrite and amountsup to 290 micromoles per milliliter of biological fluid do not interferewith test results. Preferably, 72.4 micromoles of sodium nitrite permilliliter of biological test fluids are used.

The preferred buffer solution contains ammediol hydrochloride as thebuffer, and the pH is adjusted to the required level by the addition ofadditional mineral acid, such as hydrochloric acid.

The dye former used in the process of this invention is a mineral acidsolution of 8-hydroxy quinoline sulfate. The acid also serves to stopthe enzymatic reaction so that the amount of cleavage product formedwithin a specified period of time can be measured. In order to stabilizethe final colored product being measured it has been found necessary toinclude a watersoluble, nonionic emulsifier. Among the emulsifiers whichmay be used for this purpose, there may be mentioned ethoxylatecltrideceyl alcohol, ethoxylated oleyl alcohol and ethoxylated stearicacid. These products are sold commercially under the tradenames Lipal610, Lipal 395 and Lipal 20-OA, respectively, by Drew Chemical Co.,Inc., 522 Fifth Ave., N.Y., N.Y. Another suitable nonionic emulsifierwhich may be mentioned is octyl phenoxy polyethoxy ethanol, sold underthe tradename Triton X100 by Rohm and Haas Co., lndependence Mall West,Philadelphia, Pa.

For use in the diagnostic test of the invention, the dye formingsolution must contain from about 1 1.8 to about 29.5 micromoles,preferably about 14.8 micromoles, of S-hydroxy quinoline sulfate permilliliter of biological fluid being tested. Usually from about 1 toabout 5 percent by volume, based on the total volume of the dye formingsolution, of the emulsifier, preferably 1 percent by volume ofemulsifier, is required to stabilize the dye formed.

A most important factor in the formation of the dye isthe pH of thereaction solution during dye formation. It has been found that maximumcoupling of dye takes place at a pH of from about 1.2 to about 2,preferably at a pH of about 1.9 In order to be sure that this pH isobtained, the dye forming solution which is added to the substratereaction solution, i.e., the mineral acid solution of 8-hydroxyquinoline sulfate containing the emulsifier, must have a pH of fromabout 0.8 to about 1.3, preferably about 1.1. This pH may be achieved byusing an aqueous mineral acid solution, i.e., an aqueous hydrochloricacid solution wherein the acid is at a concentration of from about 0.1Nto 0.5N. Acid at this concentration is also sufficient to stop theenzymatic activity prior to dye formation and measurement.

The final addition of the diagnostic test is an alkaline solution whichwill impart a pH of between about 10.5 to about 12, preferably about10.5, to the final reaction mixture in order to develop the color of thedye product which is to be measured. Typically, sodium hydroxide is usedand it has been found that a minimum concentration of at least 0.05Nsodium hydroxide is needed to provide consistent absorbency readings.1n-

creasing the sodium hydroxide concentration up to about 0.25N does notinterfere with the results.

1f, in the initial substrate solution, the optional magnesium chloridehexahydrate is included, a specified amount ofethylenediaminetetraacetic acid, tetrasodium salt, must be added alongwith the alkaline solution in order to prevent the precipitation of themagnesium chloride (as magnesium hydroxide) from the final reactionmixture. Such precipitation would, of course, interfere with absorbencyreadings and destroy the accuracy and sensitivity of the test. A minimumconcentration of at least 6.5 micromoles of ethylene diaminetetraaceticacid, per micromole of magnesium chloride hexahydrate, is required tomaintain solubility. Larger quantities of ethylenediaminetetraaceticacid salt may be used, i.e. up to about times as much, with similarresults. However, since the minimum concentration is sufficient, it isgenerally preferred.

For the actual assay of this invention, only about 0.1 milliliters ofbiological fluid is required for testing. Larger quantities of testsample may, of course, be used as desired. Dilution of thesample is notnecessary. Thus, blood serum, blood plasma, urine, cerebrospinal fluidand the like biological fluids may be tested directly, according to themethod of this invention.

Generally, 0.1 milliliters of sample fluid is added to l milliliter ofthe buffered substrate reagent described above and incubated for apredetermined period of time at a specified temperature. The time andtemperature may be varied greatly, but most frequently as short a periodof time and as low a temperature as necessary to insure accurate resultsare preferred Therefore, incubation at about 37C for 10 minutes isreliable and therefore recommended. After completion of the incubationtime, 1 milliliter of the mineral acid reagent solution containingS-hydroxy quinoline sulfate and the water-soluble, nonionic emulsifieris added to the reaction mixture. After the elapse of about seconds, butno more than 10 minutes, about 5 milliliters of a 0.1N sodium hydroxidesolution (containing 0.1% ethylenediaminetetraacetic acid tetrasodiumsalt, if magnesium chloride hexahydrate is included in the substratesolution) is added to the reaction solution. The absorbency of the finalsolution is read at 570 nanometers, and this value corresponds to theamount of p-nitroaniline released during the enzymatic reaction.Concentration of p-nitroaniline is determined by reading against astandard curve prepared from absorbencies of an aqueous solutioncontaining various concentrations of free pnitroaniline. Micromoles ofp-nitroaniline obtained are converted to enzymatic activity according tothe following equation:

lU Optical density at 570 nanometers X 0.172

l.U. represents lnternational Units defined as the amount of micromolesof p-nitroaniline released per 1,000 ml. of serum in one minute.

In order to further illustrate the invention, the following examples aregiven:

EXAMPLE 1 Preparation of the Substrate SolutionL-gamma-Glutamyl-p-nitroaniline, 4.5 millimolar; glycylglycine 40millimolar; sodium nitrite 7.25 millimolar; and 10 millimolar magnesiumchloride hexahydrate. Dissolve 126.3 milligramsL-gamma-glutamyl-pnitroanilide, 290.7 milligrams glycylglycine, 50milligrams sodium nitrite and 223.7 milligrams magnesium chloridehexahydrate with constant stirring in 100 milliliters ofAmmedio1-(2-amino-2-methyl-propane-1,3- diol)-HCl buffer, 0.05M, pH 8.6at 5060C. The solution has a pH of about 8.05 at 25C.

EXAMPLE 2 Preparation of the Acid Solution of 8-Hydroxy- QuinolineSulfate Dissolve 100 milligrams of 8-hydroxy quinoline sulfate inapproximately milliliters of 0.2N hydrochloric acid. Add 1 milliliter ofLipal-6l0 and dilute to milliliters with 0.2N hydrochloric acid.

EXAMPLE 3 Preparation of the Sodium Hydroxide Solution ContainingEthylenediaminetetraacetic Acid Sodium Salt Dissolve 500 milligrams ofethylenediaminetetraacetic acid, tetrasodium salt in 100 milliliters of0.05N sodium hydroxide.

EXAMPLE 4 Determination of gamma-Glutamyl Transpeptidase in Blood SerumPlace 1 milliliter of the substrate solution of Example 1 in a test tubeand add 0.1 milliliter of blood serum. Incubate at 37C for 10 minutes.When incubation is completed, add 1 milliliter of the reagent solutionof Example 2. Wait at least 15 seconds, but no more than 10 minutes andadd 5 milliliters of the reagent solution of Example 3. Read theabsorbence of the reagent solution on a Gilford 300 N spectrophotometerat 570 nanometers. A reading of 1.033 is obtained. This is read againstthe standard curve and an amount of .177 micromoles of releasedp-nitroaniline is obtained. The concentration of gamma-Glutamyltranspeptidase in the test serum is calculated according to the formula:

1.U. OD X 0.172

l.U. 1.033 X 0.172

LU. 0.177 (or 177 milliunits per milliliter) EXAMPLE 5 Determination ofgamma-Glutamyl Transpeptidase in Urine Place 1 milliliter of thesubstrate solution of Example 1 in a test tube and add 0.1 milliliter ofurine. Incubate at 37C for 10 minutes. When incubation is completed, add1 milliliter of the reagent solution of Example 2. Wait at least 15seconds, but no more than 10 minutes and add 5 milliliters of thereagent solution of Example 3. Read the absorbence of the reagentsolution on a Gilford 300 N spectrophotometer at 570 nanometers. Areading of 0.200 is obtained. This is read against the standard curveand an amount of 0.034 micromoles of released p-nitroaniline isobtained. The concentration of gamma-glutamyl transpeptidase in the testserum is calculated according to the formula:

-LU. OD X 0.172

LU. 0.200 X 0.172

LU. 0.034 (or 34 milliunits per milliliter) I claim:

1. A colorimetric method for the determination of gamma-glutamyltranspeptidase concentrations in biological fluids which comprises:

A. incubating a sample of the biological fluid with an aqueous substratesolution, said substrate solution comprising at least about 45micromoles of L- gamma-glutamyl-p-nitroanilide per milliliter ofbiological fluid, at least 400 micromoles of glycylglycine permilliliter of biological fluid, and at least 36.2 micromoles of sodiumnitrite per milliliter of biological fluid in an aqueous solutionbuffered to maintain a pH of from about 7.9 to about 8.4;

B. Continuing the incubation of solution (A) for a predetermined periodof time at a definite temperature to release p-nitroaniline as a resultof enzymatic activity;

C. Adding to solution (B) at the end of the incubation time, an aqueousmineral acid solution containing from about 1 1.8 to about 29.5micromoles of 8-hydroxy quinoline sulfate per milliliter of biologicalfluid and from about 1 to about percent by volume, based on the totalvolume of the mineral acidsolution, of a water soluble, nonionicemulsifier, said mineral acid solution having a pH of from about 0.8 toabout 1.3;

D. Allowing from about seconds to about 10 minutes to elapse and addingto solution (C) a sufficient amount of an alkaline solution to impart afinal pH of from about 10.5 to about l2 to the reaction solution; and

E. Reading the optical density of solution (D) as a measure ofgamma-glutamyl transpeptidase concentration in the biological fluid.

2. A method according to claim 1 wherein the substrate solution of (A)is buffered to a pH ofabout 8.1 with a (Z-amino-2-methyl-propane-l,3-diol)-HC1 buffer system.

3. A method according to claim 2 wherein the mineral acid solution of(C) contains about 14.8 micromoles of 8-hydroxy quinoline sulfate permilliliter of biological fluid and has a pH of about 1.].

4. A method according to claim 3 wherein the mineral acid solution of(C) contains about 1 percent by volume, based on the total volume of themineral acid solution of ethoxylated tridecyl alcoholas the watersoluble nonionic emulsifier.

5. A method according to claim 1 wherein the substrate solution of (A)additionally contains at least 100 micromoles of magnesium chloridehexahydrate per milliliter of biological fluid, and wherein the alkalinesolution of (D) additionally contains at least 6.5 micromoles ofethylenediaminetetraacetic acid, tetrasodium salt per micromole ofmagnesium chloride hexahydrate in the substrate solution of (A).

6. A method according to claim 5 wherein the substrate solution of (A)is buffered to a pH of about 8.1 with a buffer system.

7. A method according to claim 6 wherein the mineral acid solution of(C) contains about 14.8 micro- (2-amino-2-methyl-propane-l ,3 diol)-HClmoles of S-hydroxy quinoline sulfate per milliliter of biological fluidand has a pH of about 1.1.

8. A method according to claim 7 wherein the mineral acid solution of(C) contains about 1 percent by volume, based on the total volume of themineral acid solution, of ethoxylated tridecyl alcohol as the watersoluble nonionic emulsifier.

9. A colorimetric method for the determination of gamma-glutamyltranspeptidase concentrations in biological fluids which comprises:

A. lncubatingthe sample of the biological fluid with an aqueoussubstrate solution, said substrate solution comprising about 45micromoles of L-gammaglutamyl-p-nitroanilide per milliliter ofbiological fluid, about 400 micromoles of glycylglycine per milliliterof biological fluid, about 72.4 micromoles of sodium nitrite permilliliter of biological fluid, and about micromoles of magnesiumchloride hexahydrate per milliliter of biological fluid, in an aqueoussolution buffered to maintain a pH of about 8.1 with a(2-amino-2-methyl-propane-l,3- diol)-HCl buffer system;

B. Continuing the incubation of solution (A) for about ten minutes atabout 37C to release pnitroaniline as a result of enzymatic activity;

C. Adding to solution (B), at the end of the incubation time, an aqueoushydrochloric acid solution containing about 14.8 micromoles of 8-hydroxyquinoline sulfate per milliliter of biological fluid, and about 1percent by volume, based on the total volume of the hydrochloric acidsolution, of ethoxylated tridecyl alcohol, said solution having a pH ofabout l.l',

D. Allowing from about 15 seconds to about 10 minutes to elapse andadding to solution (C) a sufficient amount of an aqueous sodiumhydroxide solution to provide a final pH of about 10.5 in the reactionsolution, said sodium hydroxide solution containing about 6.5 micromolesof ethylenediaminetetraacetic acid, tetrasodium salt per micromole 'ofmagnesium chloride hexahydrate in the substrate solution A; and

E. Reading the optical .density of solution (D) as a measure ofgamma-glutamyl transpeptidase concentration in the biological fluid.

10. A method according to claim 1 wherein the substrate solution of (A)is buffered to a pH of about 8.1 with a tris(hydroxy-methyl)aminomethane hydrochloride buffer system.

11. A method according to claim 6 wherein the substrate solution of '(A)is buffered to a pH of about 8.1 with a tris(hydroxy-methyl)aminomethane hydrochloride buffer system.

2. A method according to claim 1 wherein the substrate solution of (A)is buffered to a pH of about 8.1 with a(2-amino-2-methyl-propane-1,3-diol)-HCl buffer system.
 3. A methodaccording to claim 2 wherein the mineral acid solution of (C) containsabout 14.8 micromoles of 8-hydroxy quinoline sulfate per milliliter ofbiological fluid and has a pH of about 1.1.
 4. A method according toclaim 3 wherein the mineral acid solution of (C) contains about 1percent by volume, based on the total volume of the mineral acidsolution of ethoxylated tridecyl alcohol as the water soluble nonionicemulsifier.
 5. A method according to claim 1 wherein the substratesolution of (A) additionally contains at least 100 micromoles ofmagnesium chloride hexahydrate per milliliter of biological fluid, andwherein the alkaline solution of (D) additionally contains at least 6.5micromoles of ethylenediaminetetraacetic acid, tetrasodium salt permicromole of magnesium chloride hexahydrate in the substrate solution of(A).
 6. A method according to claim 5 wherein the substrate solution of(A) is buffered to a pH of about 8.1 with a(2-amino-2-methyl-propane-1,3-diol)-HCl buffer system.
 7. A methodaccording to claim 6 wherein the mineral acid solution of (C) containsabout 14.8 micromoles of 8-hydroxy quinoline sulfate per milliliter ofbiological fluid and has a pH of about 1.1.
 8. A method according toclaim 7 wherein the mineral acid solution of (C) contains about 1percent by volume, based on the total volume of the mineral acidsolution, of ethoxylated tridecyl alcohol as the water soluble nonionicemulsifier.
 9. A colorimetric method for the determination ofgamma-glutamyl transpeptidase concentrations in biological fluids whichcomprises: A. Incubating the sample of the biological fluid with anaqueous substrate solution, said substrate solution comprising about 45micromoles of L-gamma-glutamyl-p-nitroanilide per milliliter ofbiological fluid, about 400 micromoles of glycylglycine per milliliterof biological fluid, about 72.4 micromoles of sodium nitrite permilliliter of biological fluid, and about 100 micromoles of magnesiumchloride hexahydrate per milliliter of biological fluid, in an aqueoussolution buffered to maintain a pH of about 8.1 with a(2-amino-2-methyl-propane-1,3-diol)-HCl buffer system; B. Continuing theincubation of solution (A) for about ten minutes at about 37*C torelease p-nitroaniline as a result of enzymatic activity; C. Adding tosolution (B), at the end of the incubation time, an aqueous hydrochloricacid solution containing about 14.8 micromoles of 8-hydroxy quinolinesulfate per milliliter of biological fluid, and about 1 percent byvolume, based on the total volume of the hydrochloric acid solution, ofethoxylated tridecyl alcohol, said solution having a pH of about 1.1; D.Allowing from about 15 seconds to about 10 minutes to elapse and addingto solution (C) a sufficient amount of an aqueous sodium hydroxidesolution to provide a final pH of about 10.5 in the reaction solution,said sodium hydroxide solution containing about 6.5 micromoles ofethylenediaminetetraacetic acid, tetrasodium salt per micromole ofmagnesium chloride hexahydrate in the substrate solution A; and E.Reading the optical density of solution (D) as a measure ofgamma-glutamyl transpeptidase concentration in the biological fluid. 10.A method according to claim 1 wherein the substrate solution of (A) isbuffered to a pH of about 8.1 with a tris(hydroxy-methyl)amino methanehydrochloride buffer system.
 11. A method according to claim 6 whereinthe substrate solution of (A) is buffered to a pH of aBout 8.1 with atris(hydroxy-methyl)amino methane hydrochloride buffer system.